Compositions for RNA interference and methods of use thereof

ABSTRACT

The present invention provides compositions for RNA interference and methods of use thereof. In particular, the invention provides single-stranded small interfering RNAs. Functional and genomic and proteomic methods are featured. Therapeutic methods are also featured.

RELATED APPLICATIONS

[0001] This application claims the benefit of copending U.S. provisionalpatent application Ser. No. 60/401,902 entitled “5′ Phosphorylated,Single-Stranded siRNAs that Trigger RNA Interference”, filed Aug. 7,2002, and copending U.S. provisional patent application Ser. No.60/408,786 entitled “Compositions for RNA interference and methods ofuse thereof”, filed Sep. 5, 2002. The entire contents of theabove-referenced applications are incorporated herein by this reference.

GOVERNMENT RIGHTS

[0002] This invention was made at least in part with government supportunder grant no. R01 GM62862-01 awarded by the National Institutes ofHealth. The government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] In diverse eukaryotes, double-stranded RNA (dsRNA) triggers thedestruction of mRNA sharing sequence with the double-strand (Hutvágneret al. (2002) Curr. Opin. Genet. Dev. 12:225-232; Hannon (2002) Nature418:244-251). In animals and basal eukaryotes, this process is calledRNA interference (RNAi) (Fire et al. (1998) Nature 391:806-811). Thereis now wide agreement that RNAi is initiated by the conversion of dsRNAinto 21-23 nt fragments by the multi-domain RNase III enzyme, Dicer(Bernstein et al. (2001) Nature 409:363-366; Billy et al. (2001) Proc.Natl. Acad. Sci. USA 98:14428-14433; Grishok et al. (2001) Cell106:23-34; Ketting et al. (2001) Genes Dev. 15:2654-2659; Knight et al.(2001) Science 293:2269-2271; and Martens et al. (2002) Cell13:445-453). These short RNAs are known as small interfering RNAs(siRNAs), and they direct the degradation of target RNAs complementaryto the siRNA sequence (Zamore et al. (2000) Cell 101:25-33; Elbashir etal. (2001) Nature 411:494-498; Elbashir et al. (2001) Genes Dev.15:188-200; Elbashir et al. (2001) EMBO J. 20:6877-6888; Nykänen et al.(2001) Cell 107:309-321; and Elbashir et al. (2002) Clin. Pharmacol.26:199-213). In addition to its role in initiating RNAi, Dicer alsocleaves ˜70 nt precursor RNA stem-loop structures into single-stranded21-23 nt RNAs known as microRNAs (miRNAs; Grishok et al. (supra);Hutvágner et al. (2001) Science 293:834-838; Ketting et al. (supra); andReinhart et al. (2002) Genes Dev. 16:1616-1626). Like siRNAs, miRNAsbear 5′ monophosphate and 3′ hydroxyl groups, the signatures of RNaseIII cleavage products (Elbashir et al. (supra); Hutvágner et al.(supra). miRNAs are hypothesized to function in animals as translationalrepressors (Lee et al. (1993) Cell 75:843-854; Wightman et al. (1993)Cell 75:855-862; Ha et al. (1996) Genes Dev. 10:3041-3050; Moss et al.(1997) Cell 88:637-646; Olsen et al. (1999) Dev. Biol. 216:671-680;Reinhart et al. (2000) Nature 403:901-906; Zeng et al. (2002) MolecularCell 9:1327-1333; and Seggerson et al. (2002) Dev. Biol. 243:215-225).The conversion of dsRNA into siRNAs requires additional proteinco-factors that may recruit the dsRNA to Dicer or stabilize the siRNAproducts (Tabara et al. (1999) Cell 99:123-132; Grishok et al. (supra);Hammond et al. (2001) Science 293:1146-1150; and Tabara et al. (2002)Cell 109:861-871).

SUMMARY OF THE INVENTION

[0004] In Drosophila, several features of small interfering RNA (siRNA)structure are reported to be essential for RNA interference (RNAi). Inparticular, it is reported that siRNAs must be double stranded to beeffective in mediating RNAi. Moreover, 5′ phosphates and 3′ hydroxylsare reported to be essential for RNA interference (RNAi). The presentinvention is based, at least in part, on the surprising discovery thatsingle-stranded siRNAs are efficient in mediating target-specific RNAi.Moreover, in both Drosophila and mammalian cell extracts, as well as invivo in human HeLa cells, a 5′ phosphate is required for siRNA function.In contrast, there is no evidence in flies or humans for a role in RNAifor the siRNA 3′ hydroxyl group. The present invention is further based,in part, on the premise that in both flies and mammals, each siRNAdirects endonucleolytic cleavage of the target RNA at a single site. Itcan be concluded that the underlying mechanism of RNAi is conservedbetween flies and mammals and that RNA-dependent RNA polymerases are notrequired for RNAi in these organisms.

[0005] Accordingly, the present invention provides compositions for RNAinterference and methods of use thereof. In particular, the inventionprovides single-stranded small interfering RNAs (ss-siRNAs) formediating RNAi in vitro and in vivo. Methods for using saidsingle-stranded small interfering RNAs are also provided. In particular,functional and genomic and proteomic methods are featured. Therapeuticmethods are also featured.

[0006] The ss-siRNA molecules of the invention are particularly usefulas reagents for RNA interference and have improved efficacy and are moreeconomical to make than prior art siRNA agents.

[0007] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1. Endonucleolytic cleavage model for RNAi in Drosophila. Themodel postulates that dsRNA is converted to siRNA by the ATP-dependentendoribonuclease Dicer, but the models differ as to the subsequentfunction of siRNAs. siRNAs are proposed to be incorporated into anendonuclease complex distinct from Dicer, the RISC. Assembly of the RISCis proposed to be ATP-dependent, whereas endonucleolytic cleavage of thetarget RNA is postulated to require no high energy cofactors.

[0009]FIG. 2. RNAs used in this study. (A) Photinus pyralis (firefly)luciferase and let-7 siRNAs used in this study. The guide strand(antisense strand) is shown 5′-to-3′ as the upper strand of each siRNA.Single-stranded siRNAs used in FIGS. 4, 5, and 6 correspond to theindicated guide strands. ddC, dideoxy Cytosine; AM, amino modifier. thefirst 10 siRNA duplexes correspond to the firefly luciferase sequence;the last two siRNA duplexes correspond to the let-7 sequence. (B) Aschematic representation of the chimeric target RNA, indicating therelative positions of firefly luciferase sequences and sequencescomplementary to the let-7 miRNA found naturally in HeLa cells.

[0010]FIG. 3. The siRNA 5′ phosphate group is required forsiRNA-directed target cleavage in HeLa S100 extracts. (A) RNAi in vitroin human HeLa cell S100 extract. At left, a time course of in vitro RNAifor a standard siRNA; at right, for an siRNA duplex bearing a 5′ methoxyguide strand. The asterisk indicates the position of a 5′ cleavageproduct catalyzed by an endogenous, human let-7-programmed RISC complex,which cleaves this target RNA within a let-7 complementary sequencelocated near the 3′ end of the RNA (Hutvágner and Zamore, 2002a). Thiscleavage product serves as an internal control. (B) Phosphorylationstatus of the guide strand of an siRNA duplex upon incubation in HeLaS100. An siRNA duplex containing a guide strand 3′-end-labeled with a-₃₂P cordycepin (3′ deoxyadenosine triphosphate) was incubated in astandard HeLa S100 RNAi reaction, then analyzed on a 15% sequencing gel.Phosphorylation accelerates the gel mobility of the labeled siRNAstrand, because it adds two additional negative charges. Theradiolabeled RNA is 3′ deoxy; therefore, we infer that the addedphosphate is on the 5′ end.

[0011]FIG. 4. The siRNA 3′ hydroxyl is dispensable for siRNA-directedtarget cleavage in Drosophila and human cell extracts. (A) 3′-blockedsiRNAs trigger RNAi in Drosophila embryo lysates with the sameefficiency as 3′-hydroxyl-containing siRNAs. ddC, 2′,3′ dideoxy C; AM,amino modifier. (B) 3′-blocked siRNAs trigger RNAi in HeLa S100 extractswith the same efficiency as standard, 3′-hydroxyl-containing siRNAs. Anover-exposure of the region of the gel containing the 5′ cleavageproduct is shown in the lower panel. The asterisk marks the internalcontrol 5′ cleavage product described in FIG. 3.

[0012]FIG. 5. Single-stranded siRNA guides target cleavage in Drosophilaembryo lysates. (A) Single-stranded siRNAs with the sequence of themiRNA let-7 triggered target cleavage in Drosophila embryo lysate, butonly if the 5′ end was pre-phosphorylated. (B) Single-stranded siRNAscomplementary to firefly luciferase sequence triggered target cleavagein Drosophila embryo lysate, even if the 3′ end was blocked (2′,3′ddC).No target cleavage was observed using an siRNA with a 5′ methoxy group.(C) Rate of degradation of single-stranded siRNA in the Drosophilaembryo lysate. siRNA single-strands were 3′ end-labeled with a—₃₂Pcordycepin and their stability measured with (filled circles) or without(open squares) a 5′ phosphate. The curves represent the best-fit to asingle exponential, consistent with pseudo first-order kinetics forsingle-stranded siRNA decay. The difference in rates is 1.4-fold (withversus without a 5′ phosphate).

[0013]FIG. 6. A 5′ phosphate, but not a 3′ hydroxyl is required forsingle-stranded antisense siRNAs to trigger RNAi in HeLa S100 extract.(A) Single-stranded siRNA triggered target cleavage in HeLa S100, evenif the 3′ end of the siRNA was blocked (2′,3′ dideoxy). (B) Blocking the5′ end of the siRNA with a methoxy group eliminated the ability of thesingle-stranded RNA to trigger RNAi. The asterisk marks the control 5′cleavage product described in FIG. 3.

[0014]FIG. 7. A 5′ phosphate, but not a 3′ hydroxyl is required forsiRNA duplexes to trigger RNAi in vivo in cultured human HeLa cells. (A)siRNA duplexes were examined for their ability to silence the Photinuspyralis (Pp; firefly) luciferase target reporter, relative to theRenilla reniformis (Rr) luciferase control reporter. ddC, 2′,3′ dideoxyC; AM, amino modifier. (B) Relative efficacy at limiting siRNAconcentrations for siRNA duplexes with guide strands bearing eitherhydroxy (symbols connected by solid lines) or ddC (symbols connected bydashed line) 3′ termini. Data are the average±standard deviation forthree trials.

[0015]FIG. 8. Single-stranded siRNA triggers gene silencing in HeLacells. (A) Single-stranded siRNA silencing as a function of siRNAconcentration. (B) Blocking the 5′ end of single-stranded siRNAsprevented their triggering target gene silencing. Gray bars indicate theaverage±standard deviation for three trials. Un, siRNA unrelated insequence to the target RNA; sp, specific siRNA corresponding to thetarget RNA; s, sense strand; as, anti-sense strand.

DETAILED DESCRIPTION OF THE INVENTION

[0016] In one aspect, the invention provides an isolated,single-stranded small interfering molecule (ss-siRNA), wherein thesequence of said ss-siRNA molecule is sufficiently complementary to atarget mRNA sequence to direct target-specific RNA interference (RNAi)and wherein the 5′ nucleotide is 5′ phosphorylated or is capable ofbeing 5′ phosphorylated in situ or in vivo.

[0017] In one embodiment, the ss-siRNA is sufficiently complementary toa target mRNA, said target mRNA specifying the amino acid sequence of acellular protein. In another embodiment, the ss-siRNA is sufficientlycomplementary to a target mRNA, said target mRNA specifying the aminoacid sequence of a viral protein. Additionally or alternatively, thess-siRNA can be modified such that the ss-siRNA has increased in situ orin vivo stability as compared to a corresponding unmodified ss-siRNA.Such a modified ss-siRNA can be modified by the substitution of at leastone nucleotide with a modified nucleotide, which can be a sugar-modifiednucleotide. The sugar-modified nucleotide can have a 2′—OH replaced by amoiety selected from the group consisting of H, OR, R, halo, SH, SR¹,NH₂, NHR, NR₂ and CN, wherein R is C₁-C₆ alkyl, alkenyl or alkynyl andhalo is F, Cl, Br or I.

[0018] The sugar-modified nucleotide can be a 3′ most nucleotide. In oneembodiment, the 3′ most nucleotide has a 2′—OH replaced by a moietyselected from the group consisting of H, OR, R, halo, SH, SR¹, NH₂, NHR,NR₂ and CN, wherein R is C₁-C₆ alkyl, alkenyl or alkynyl and halo is F,Cl, Br or I. In another embodiment, the 3′ most nucleotide has a 3′—OHreplaced by a moiety selected from the group consisting of H, OR, R,halo, SH, SR¹, NH₂, NHR, NR₂ and CN, wherein R is C₁-C₆ alkyl, alkenylor alkynyl and halo is F, Cl, Br or I.

[0019] In yet another embodiment, the modified nucleotide is abackbone-modified nucleotide. In this embodiment, the backbone-modifiednucleotide may contain a phosphorothioate group.

[0020] In yet another embodiment, the ss-siRNA of the invention caninclude between about 10 and 50 nucleotides, preferably between about 15and 45 nucleotides, more preferably, between about 19 and 40nucleotides.

[0021] In a further embodiment, the ss-siRNA of of the invention ischemically synthesized.

[0022] In another aspect, the invention provides a transgene thatencodes any of the ss-siRNA described herein. In yet another aspect, theinvention provides composition including an ss-siRNA molecule of theinvention, and a pharmaceutically acceptable carrier.

[0023] In still yet another aspect, the invention provides a method ofactivating target-specific RNA interference (RNAi) in a cell. The methodincludes the step of introducing into said cell a single-stranded smallinterfering RNA molecule (ss-siRNA), wherein the sequence of saidss-siRNA molecule is sufficiently complementary to a target mRNAsequence to direct target-specific RNA interference (RNAi) and whereinthe 5′ nucleotide is 5′ phosphorylated or is capable of being 5′phosphorylated in situ or in vivo, said ss-siRNA being introduced in anamount sufficient for degradation of the target mRNA to occur, therebyactivating target-specific RNAi in the cell.

[0024] In one embodiment of this method, the ss-siRNA is introduced intothe cell by contacting the cell with the ss-siRNA. In another, thess-siRNA is introduced into the cell by contacting the cell with acomposition comprising the ss-siRNA and a lipophillic carrier. In yetanother, the ss-siRNA is introduced into the cell by transfecting orinfecting the cell with a vector comprising nucleic acid sequencescapable of producing the ss-siRNA when transcribed in situ. In yetanother, the ss-siRNA is introduced into the cell by injecting into thecell a vector comprising nucleic acid sequences capable of producing thess-siRNA when transcribed in situ. Such a vector includes transgenenucleic acid sequences in yet another embodiment of the invention.

[0025] In another embodiment of the method, the target mRNA specifiesthe amino acid sequence of a protein involved or predicted to beinvolved in a human disease or disorder.

[0026] In another aspect, the invention provides a cell obtained by amethod of the invention. In various embodiments, the cell can be ofmammalian origin, of murine origin, or of human origin. In anotherembodiment, cell is an embryonic stem cell. The invention furtherprovides an organism derived from an embryonic stem cell obtained by amethod of the invention.

[0027] In yet another aspect, the invention provides a method ofactivating target-specific RNA interference (RNAi) in an organism. Themethod includes the step of administering to said organism asingle-stranded small interfering RNA molecule (ss-siRNA), wherein thesequence of said ss-siRNA molecule is sufficiently complementary to atarget mRNA sequence to direct target-specific RNA interference (RNAi)and wherein the 5′ nucleotide is 5′ phosphorylated or is capable ofbeing 5′ phosphorylated in situ or in vivo, said ss-siRNA beingadministered in an amount sufficient for degradation of the target mRNAto occur, thereby activating target-specific RNAi in the organism.

[0028] In one embodiment, the the ss-siRNA is administered by anintravenous or intraperitoneal route. In another, the target mRNAspecifies the amino acid sequence of a protein involved or predicted tobe involved in a human disease or disorder.

[0029] The invention further provides an organism obtained by thismethod. In various embodiments, the organism can be of mammalian origin,murine origin or human origin. In any of these embodiments, a furtherembodiment may include organisms wherein the target mRNA specifies theamino acid sequence of a protein involved or predicted to be involved ina human disease or disorder. In a preferred embodiment, degradation ofthe target mRNA produces a loss-of-function phenotype.

[0030] In any of the methods of the invention, a further embodimentincludes a method wherein degradation of the target mRNA is such thatthe protein specified by said target mRNA is decreased by at least 10%.

[0031] In yet another aspect, the invention provides a method oftreating a disease or disorder associated with the activity of a proteinspecified by a target mRNA in a subject. The method includesadministering to said subject a single-stranded small interfering RNAmolecule (ss-siRNA), wherein the sequence of said ss-siRNA molecule issufficiently complementary to the target mRNA sequence to directtarget-specific RNA interference (RNAi) and wherein the 5′ nucleotide is5′ phosphorylated or is capable of being 5′ phosphorylated in situ or invivo, said ss-siRNA being administered in an amount sufficient fordegradation of the target mRNA to occur, thereby treating the disease ordisorder associated with the protein.

[0032] In still yet another aspect, the invention provides a method forderiving information about the function of a gene in a cell or organism.The method includes the steps of: (a) introducing into said cell ororganism a single-stranded small interfering RNA molecule (ss-siRNA),wherein the sequence of said ss-siRNA molecule is sufficientlycomplementary to a target mRNA sequence to direct target-specific RNAinterference (RNAi) and wherein the 5′ nucleotide is 5′ phosphorylatedor is capable of being 5′ phosphorylated in situ or in vivo; (b)maintaining the cell or organism under conditions such thattarget-specific RNAi can occur; (c) determining a characteristic orproperty of said cell or organism; and (d) comparing said characteristicor property to a suitable control, the comparison yielding informationabout the function of the gene.

[0033] In yet another aspect, the invention provides a method ofvalidating a candidate protein as a suitable target for drug discovery.The method includes the steps of: (a) introducing into a cell ororganism a single-stranded small interfering RNA molecule (ss-siRNA),wherein the sequence of said ss-siRNA molecule is sufficientlycomplementary to a target mRNA sequence to direct target-specific RNAinterference (RNAi) and wherein the 5′ nucleotide is 5′ phosphorylatedor is capable of being 5′ phosphorylated in situ or in vivo, said targetmRNA specifying the amino acid sequence of the candidate protein; (b)maintaining the cell or organism under conditions such thattarget-specific RNAi can occur; (c) determining a characteristic orproperty of said cell or organism; and (d) comparing said characteristicor property to a suitable control, the comparison yielding informationabout whether the candidate protein is a suitable target for drugdiscovery.

[0034] In another aspect, the invention provides a kit comprisingreagents for activating target-specific RNA interference (RNAi) in acell or organism. The kit may include: (a) an isolated, single-strandedsmall interfering (ss-siRNA) molecule, wherein the sequence of saidss-siRNA molecule is sufficiently complementary to a target mRNAsequence to direct target-specific RNA interference (RNAi) and whereinthe 5′ nucleotide is 5′ phosphorylated or is capable of being 5′phosphorylated in situ or in vivo; and (b) instructions for use.

[0035] So that the invention may be more readily understood, certainterms are first defined.

[0036] The term “nucleoside” refers to a molecule having a purine orpyrimidine base covalently linked to a ribose or deoxyribose sugar.Exemplary nucleosides include adenosine, guanosine, cytidine, uridineand thymidine. The term “nucleotide” refers to a nucleoside having oneor more phosphate groups joined in ester linkages to the sugar moiety.Exemplary nucleotides include nucleoside monophosphates, diphosphatesand triphosphates. The terms “polynucleotide” and “nucleic acidmolecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester linkage between 5′ and3′ carbon atoms.

[0037] The term “RNA” or “RNA molecule” or “ribonucleic acid molecule”refers to a polymer of ribonucleotides. The term “DNA” or “DNA molecule”or deoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., byDNA replication or transcription of DNA, respectively). RNA can bepost-transcriptionally modified. DNA and RNA can also be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNAthat specifies the amino acid sequence of one or more polypeptidechains. This information is translated during protein synthesis whenribosomes bind to the mRNA.

[0038] As used herein, the term “small interfering RNA” (“siRNA”) (alsoreferred to in the art as “short interfering RNAs”) refers to an RNA (orRNA analog) comprising between about 10-50 nucleotides (or nucleotideanalogs) which is capable of directing or mediating RNA interference.

[0039] The term “nucleotide analog” or “altered nucleotide” or “modifiednucleotide” refers to a non-standard nucleotide, including non-naturallyoccurring ribonucleotides or deoxyribonucleotides. Preferred nucleotideanalogs are modified at any position so as to alter certain chemicalproperties of the nucleotide yet retain the ability of the nucleotideanalog to perform its intended function. Examples of positions of thenucleotide which may be derivitized include the 5 position, e.g.,5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine,5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyluridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromoguanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotideanalogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- andN-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwiseknown in the art) nucleotides; and other heterocyclically modifiednucleotide analogs such as those described in Herdewijn, AntisenseNucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

[0040] Nucleotide analogs may also comprise modifications to the sugarportion of the nucleotides. For example the 2′ OH-group may be replacedby a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂,COOR, or OR, wherein R is substituted or unsubstituted C₁-C₆ alkyl,alkenyl, alkynyl, aryl, etc. Other possible modifications include thosedescribed in U.S. Pat. Nos. 5,858,988, and 6,291,438.

[0041] The phosphate group of the nucleotide may also be modified, e.g.,by substituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct.11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)preferably decrease the rate of hydrolysis of, for example,polynucleotides comprising said analogs in vivo or in vitro.

[0042] The term “oligonucleotide” refers to a short polymer ofnucleotides and/or nucleotide analogs. The term “RNA analog” refers toan polynucleotide (e.g., a chemically synthesized polynucleotide) havingat least one altered or modified nucleotide as compared to acorresponding unaltered or unmodified RNA but retaining the same orsimilar nature or function as the corresponding unaltered or unmodifiedRNA. As discussed above, the oligonucleotides may be linked withlinkages which result in a lower rate of hydrolysis of the RNA analog ascompared to an RNA molecule with phosphodiester linkages. For example,the nucleotides of the analog may comprise methylenediol, ethylene diol,oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate,phophoroamidate, and/or phosphorothioate linkages. Preferred RNAanalogues include sugar- and/or backbone-modified ribonucleotides and/ordeoxyribonucleotides. Such alterations or modifications can furtherinclude addition of non-nucleotide material, such as to the end(s) ofthe RNA or internally (at one or more nucleotides of the RNA). An RNAanalog need only be sufficiently similar to natural RNA that it has theability to mediate (mediates) RNA interference.

[0043] As used herein, the term “RNA interference” (“RNAi”) refers to aselective intracellular degradation of RNA. RNAi occurs in cellsnaturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAiproceeds via fragments cleaved from free dsRNA which direct thedegradative mechanism to other similar RNA sequences. Alternatively,RNAi can be initiated by the hand of man, for example, to silence theexpression of target genes.

[0044] A ss-siRNA having a “sequence sufficiently complementary to atarget mRNA sequence to direct target-specific RNA interference (RNAi)”means that the ss-siRNA has a sequence sufficient to trigger thedestruction of the target mRNA by the RNAi machinery or process.

[0045] The term “phosphorylated” means that at least one phosphate groupis attached to a chemical (e.g., organic) compound. Phosphate groups canbe attached, for example, to proteins or to sugar moieties via thefollowing reaction: free hydroxyl group+phosphate donor→phosphate esterlinkage. The term “5′ phosphorylated” is used to describe, for example,polynucleotides or oligonucleotides having a phosphate group attachedvia ester linkage to the C5 hydroxyl of the 5′ sugar (e.g., the 5′ribose or deoxyribose, or an analog of same). Mono-, di-, andtriphosphates are common. Also intended to be included within the scopeof the instant invention are phosphate group analogs which function inthe same or similar manner as the mono-, di-, or triphosphate groupsfound in nature (see e.g., exemplified analogs.)

[0046] As used herein, the term “isolated RNA” (e.g., “isolated ssRNA”,“isolated siRNA” or “isolated ss-siRNA”) refers to RNA molecules whichare substantially free of other cellular material, or culture mediumwhen produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.

[0047] The term “in vitro” has its art recognized meaning, e.g.,involving purified reagents or extracts, e.g., cell extracts. The term“in vivo” alos has its art recognized meaning, e.g., involving livingcells, e.g., immortalized cells, primary cells, cell lines, and/or cellsin an organism.

[0048] A gene “involved” in a disorder includes a gene, the normal oraberrant expression or function of which effects or causes a disease ordisorder or at least one symptom of said disease or disorder

[0049] The phrase “examining the function of a gene in a cell ororganism” refers to examining or studying the expression, activity,function or phenotype arising therefrom.

[0050] Various methodologies of the instant invention include step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control”, referred to interchangeably herein as an“appropriate control”. A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to introducing a ss-siRNA of the invention into a cell ororganism. In another embodiment, a “suitable control” or “appropriatecontrol” is a value, level, feature, characteristic, property, etc.determined in a cell or organism, e.g., a control or normal cell ororganism, exhibiting, for example, normal traits. In yet anotherembodiment, a “suitable control” or “appropriate control” is apredefined value, level, feature, characteristic, property, etc.

[0051] Various aspects of the invention are described in further detailin the following subsections.

[0052] I. RNA Molecules

[0053] The present invention features “single-stranded small interferingRNA molecules” (“ss-siRNA molecules” or “ss-siRNA”), methods of makingsaid ss-siRNA molecules and methods (e.g., research and/or therapeuticmethods) for using said ss-siRNA molecules. Preferably, the ss-siRNAmolecule has a length from about 10-50 or more nucleotides. Morepreferably, the ss-siRNA molecule has a length from about 15-45nucleotides. Even more preferably, the ss-siRNA molecule has a lengthfrom about 19-40 nucleotides. The ss-siRNA molecules of the inventionfurther have a sequence that is “sufficiently complementary” to a targetmRNA sequence to direct target-specific RNA interference (RNAi), asdefined herein, i.e., the ss-siRNA has a sequence sufficient to triggerthe destruction of the target mRNA by the RNAi machinery or process. Thess-siRNA molecule can be designed such that every residue iscomplementary to a residue in the target molecule. Alternatively,substitutions can be made within the molecule to increase stabilityand/or enhance processing activity of said molecule. Substitutions canbe made within the strand or can be made to residues a the ends of thestrand. The 5′-terminus is, most preferably, phosphorylated (i.e.,comprises a phosphate, diphosphate, or triphosphate group). Contrary toprevious findings, however, that the 3′ end of an siRNA be a hydroxylgroup in order to facilitate RNAi, the present inventors havedemonstrated that there is no requirement for a 3′ hydroxyl group whenthe active agent is a ss-siRNA molecule. Accordingly, the inventionfeatures, in particular, ss-siRNA molecules wherein the 3′ end (i.e., C3of the 3′ sugar) lacks a hydroxyl group (i.e., ss-siRNA moleculeslacking a 3′ hydroxyl or C3 hydroxyl on the 3′ sugar (e.g., ribose ordeoxyribose).

[0054] The target RNA cleavage reaction guided by siRNAs (e.g., byss-siRNAs) is highly sequence specific. In general, siRNA containing anucleotide sequences identical to a portion of the target gene arepreferred for inhibition. However, 100% sequence identity between thesiRNA and the target gene is not required to practice the presentinvention. Thus the invention has the advantage of being able totolerate sequence variations that might be expected due to geneticmutation, strain polymorphism, or evolutionary divergence. For example,siRNA sequences with insertions, deletions, and single point mutationsrelative to the target sequence have also been found to be effective forinhibition. Alternatively, siRNA sequences with nucleotide analogsubstitutions or insertions can be effective for inhibition.

[0055] Moreover, not all positions of a siRNA contribute equally totarget recognition. Mismatches in the center of the siRNA are mostcritical and essentially abolish target RNA cleavage. In contrast, the3′ nucleotides of the siRNA do not contribute significantly tospecificity of the target recognition. In particular, residues 3′ of thesiRNA sequence which is complementary to the target RNA (e.g., the guidesequence) are not critical for target RNA cleavage.

[0056] Sequence identity may determined by sequence comparison andalignment algorithms known in the art. To determine the percent identityof two nucleic acid sequences (or of two amino acid sequences), thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

[0057] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

[0058] In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

[0059] Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between thess-siRNA and the portion of the target gene is preferred. Alternatively,the ss-siRNA may be defined functionally as a nucleotide sequence (oroligonucleotide sequence) that is capable of hybridizing with a portionof the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed bywashing). Additional preferred hybridization conditions includehybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamidefollowed by washing at 70° C in 0.3×SSC or hybridization at 70° C. in4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in1×SSC. The hybridization temperature for hybrids anticipated to be lessthan 50 base pairs in length should be 5-10° C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49base pairs in length, Tm(° C.)=81.5+16.6(log10[Na+])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] isthe concentration of sodium ions in the hybridization buffer ([Na+] for1×SSC=0.165 M). Additional examples of stringency conditions forpolynucleotide hybridization are provided in Sambrook, J., E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters9 and 11, and Current Protocols in Molecular Biology, 1995, F. M.Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4, incorporated herein by reference. The length of the identicalnucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25,27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

[0060] In a preferred aspect, the RNA molecules of the present inventionare modified to improve stability in serum or in growth medium for cellcultures. In order to enhance the stability, the 3′-residues may bestabilized against degradation, e.g., they may be selected such thatthey consist of purine nucleotides, particularly adenosine or guanosinenucleotides. Alternatively, substitution of pyrimidine nucleotides bymodified analogues, e.g., substitution of uridine by 2′-deoxythymidineis tolerated and does not affect the efficiency of RNA interference. Forexample, the absence of a 2′ hydroxyl may significantly enhance thenuclease resistance of the ss-siRNAs in tissue culture medium.

[0061] In an especially preferred embodiment of the present inventionthe RNA molecule may contain at least one modified nucleotide analogue.The nucleotide analogues may be located at positions where thetarget-specific activity, e.g., the RNAi mediating activity is notsubstantially effected, e.g., in a region at the 5′-end and/or the3′-end of the RNA molecule. Particularly, the ends may be stabilized byincorporating modified nucleotide analogues.

[0062] Preferred nucleotide analogues include sugar- and/orbackbone-modified ribonucleotides (i.e., include modifications to thephosphate-sugar backbone). For example, the phosphodiester linkages ofnatural RNA may be modified to include at least one of a nitrogen orsulfur heteroatom. In preferred backbone-modified ribonucleotides thephosphoester group connecting to adjacent ribonucleotides is replaced bya modified group, e.g., of phosphothioate group. In preferredsugar-modified ribonucleotides, the 2′ OH-group is replaced by a groupselected from H, OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R isC₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

[0063] Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

[0064] RNA may be produced enzymatically or by partial/total organicsynthesis, any modified nibonucleotide can be introduced by in vitroenzymatic or organic synthesis. In one embodiment, a ss-siRNA isprepared chemically. Methods of synthesizing RNA molecules are known inthe art, in particular, the chemical synthesis methods as de scribed inVerma and Eckstein (1998) Annul Rev. Biochem. 67:99-134. In anotherembodiment, a ss-siRNA is prepared enzymatically. For example, ads-siRNA can be prepared by enzymatic processing of a long ds RNA havingsufficient complementarity to the desired target mRNA. Processing oflong ds RNA can be accomplished in vitro, for example, using appropriatecellular lysates and ds-siRNAs can be subsequently purified by gelelectrophoresis or gel filtration. ds-siRNA can then be denaturedaccording to art-recognized methodologies. In an exemplary embodiment,RNA can be purified from a mixture by extraction with a solvent orresin, precipitation, electrophoresis, chromatography, or a combinationthereof. Alternatively, the RNA may be used with no or a minimum ofpurification to avoid losses due to sample processing. Alternatively,the single-stranded RNAs can also be prepared by enzymatic transcriptionfrom synthetic DNA templates or from DNA plasmids isolated fromrecombinant bacteria. Typically, phage RNA polymerases are used such asT7, T3 or SP6 RNA polymerase (Milligan and Uhlenbeck (1989) MethodsEnzymol. 180:51-62). The RNA may be dried for storage or dissolved in anaqueous solution. The solution may contain buffers or salts to inhibitannealing, and/or promote stabilization of the single strands.

[0065] In one embodiment, the target mRNA of the invention specifies theamino acid sequence of a cellular protein (e.g., a nuclear, cytoplasmic,transmembrane, or membrane-associated protein). In another embodiment,the target mRNA of the invention specifies the amino acid sequence of anextracellular protein (e.g., an extracellular matrix protein or secretedprotein). As used herein, the phrase “specifies the amino acid sequence”of a protein means that the mRNA sequence is translated into the aminoacid sequence according to the rules of the genetic code. The followingclasses of proteins are listed for illustrative purposes: developmentalproteins (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt familymembers, Pax family members, Winged helix family members, Hox familymembers, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2,BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS,FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN,NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressorproteins (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RB I, TP53,and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturasesand hydroxylases, ADP-glucose pyrophorylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextriinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hernicellulases, integrases, inulinases, invertases, isomerases,kinases, lactases, lipases, lipoxygenases, lysozymes, nopalinesynthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, and xylanases).

[0066] In a preferred aspect of the invention, the target mRNA moleculeof the invention specifies the amino acid sequence of a proteinassociated with a pathological condition. For example, the protein maybe a pathogen-associated protein (e.g., a viral protein involved inimmunosuppression of the host, replication of the pathogen, transmissionof the pathogen, or maintenance of the infection), or a host proteinwhich facilitates entry of the pathogen into the host, drug metabolismby the pathogen or host, replication or integration of the pathogen'sgenome, establishment or spread of infection in the host, or assembly ofthe next generation of pathogen. Alternatively, the protein may be atumor-associated protein or an autoimmune disease-associated protein.

[0067] In one embodiment, the target mRNA molecule of the inventionspecifies the amino acid sequence of an endogenous protein (i.e., aprotein present in the genome of a cell or organism). In anotherembodiment, the target mRNA molecule of the invention specified theamino acid sequence of a heterologous protein expressed in a recombinantcell or a genetically altered organism. In another embodiment, thetarget mRNA molecule of the invention specified the amino acid sequenceof a protein encoded by a transgene (i.e., a gene construct inserted atan ectopic site in the genome of the cell). In yet another embodiment,the target mRNA molecule of the invention specifies the amino acidsequence of a protein encoded by a pathogen genome which is capable ofinfecting a cell or an organism from which the cell is derived.

[0068] By inhibiting the expression of such proteins, valuableinformation regarding the function of said proteins and therapeuticbenefits which may be obtained from said inhibition may be obtained.

[0069] In one embodiment, ss-siRNAs are synthesized either in vivo, insitu, or in vitro. Endogenous RNA polymerase of the cell may mediatetranscription in vivo or in situ, or cloned RNA polymerase can be usedfor transcription in vivo or in vitro. For transcription from atransgene in vivo or an expression construct, a regulatory region (e.g.,promoter, enhancer, silencer, splice donor and acceptor,polyadenylation) may be used to transcribe the ss-siRNA. Inhibition maybe targeted by specific transcription in an organ, tissue, or cell type;stimulation of an environmental condition (e.g., infection, stress,temperature, chemical inducers); and/or engineering transcription at adevelopmental stage or age. A transgenic organism that expressesss-siRNA from a recombinant construct may be produced by introducing theconstruct into a zygote, an embryonic stem cell, or another multipotentcell derived from the appropriate organism.

[0070] II. Methods of Introducing RNAs, Vectors, and Host Cells

[0071] Physical methods of introducing nucleic acids include injectionof a solution containing the RNA, bombardment by particles covered bythe RNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, inhibit annealing of single strands,stabilize the single strands, or other-wise increase inhibition of thetarget gene.

[0072] RNA may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing a cell or organism in a solutioncontaining the RNA. Vascular or extravascular circulation, the blood orlymph system, and the cerebrospinal fluid are sites where the RNA may beintroduced.

[0073] The cell with the target gene may be derived from or contained inany organism. The organism may a plant, animal, protozoan, bacterium,virus, or fungus. The plant may be a monocot, dicot or gymnosperm; theanimal may be a vertebrate or invertebrate. Preferred microbes are thoseused in agriculture or by industry, and those that are pathogenic forplants or animals. Fungi include organisms in both the mold and yeastmorphologies. Plants include arabidopsis; field crops (e.g., alfalfa,barley, bean, com, cotton, flax, pea, rape, nice, rye, safflower,sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops (e.g.,asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery,cucumber, eggplant, lettuce, onion, pepper, potato, pumpkin, radish,spinach, squash, taro, tomato, and zucchini); fruit and nut crops (e.g.,almond, apple, apricot, banana, black-berry, blueberry, cacao, cherry,coconut, cranberry, date, faJoa, filbert, grape, grapefr-uit, guava,kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passionfruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry,strawberry, tangerine, walnut, and watermelon); and ornamentals (e.g.,alder, ash, aspen, azalea, birch, boxwood, camellia, carnation,chrysanthemum, elm, fir, ivy, jasmine, juniper, oak, palm, poplar, pine,redwood, rhododendron, rose, and rubber). Examples of vertebrate animalsinclude fish, mammal, cattle, goat, pig, sheep, rodent, hamster, mouse,rat, primate, and human; invertebrate animals include nematodes, otherworms, drosophila, and other insects.

[0074] The cell having the target gene may be from the germ line orsomatic, totipotent or pluripotent, dividing or non-dividing, parenchymaor epithelium, immortalized or transformed, or the like. The cell may bea stem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

[0075] Depending on the particular target gene and the dose of doublestranded RNA material delivered, this process may provide partial orcomplete loss of function for the target gene. A reduction or loss ofgene expression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or moreof targeted cells is exemplary. Inhibition of gene expression refers tothe absence (or observable decrease) in the level of protein and/or mRNAproduct from a target gene. Specificity refers to the ability to inhibitthe target gene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS).

[0076] For RNA-mediated inhibition in a cell line or whole organism,gene expression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of ss-siRNA may result in inhibitionin a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%,or 95% of targeted cells). Quantitation of gene expression in a cell mayshow similar amounts of inhibition at the level of accumulation oftarget mRNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell; mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

[0077] The RNA may be introduced in an amount which allows delivery ofat least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500or 1000 copies per cell) of material may yield more effectiveinhibition; lower doses may also be useful for specific applications.

[0078] III. Methods of Treatment:

[0079] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with aberrant or unwantedtarget gene expression or activity. “Treatment”, or “treating” as usedherein, is defined as the application or administration of a therapeuticagent (e.g., a ss-siRNA or vector or transgene encoding same) to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disease ordisorder, a symptom of disease or disorder or a predisposition toward adisease or disorder, with the purpose to cure, heal, alleviate, relieve,alter, remedy, ameliorate, improve or affect the disease or disorder,the symptoms of the disease or disorder, or the predisposition towarddisease.

[0080] With regards to both prophylactic and therapeutic methods oftreatment, such treatments may be specifically tailored or modified,based on knowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

[0081] 1. Prophylactic Methods

[0082] In one aspect, the invention provides a method for preventing ina subject, a disease or condition associated with an aberrant orunwanted target gene expression or activity, by administering to thesubject a therapeutic agent (e.g., a ss-siRNA or vector or transgeneencoding same). Subjects at risk for a disease which is caused orcontributed to by aberrant or unwanted target gene expression oractivity can be identified by, for example, any or a combination ofdiagnostic or prognostic assays as described herein. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the target gene aberrancy, such that a disease ordisorder is prevented or, alternatively, delayed in its progression.Depending on the type of target gene aberrancy, for example, a targetgene, target gene agonist or target gene antagonist agent can be usedfor treating the subject. The appropriate agent can be determined basedon screening assays described herein.

[0083] 2. Therapeutic Methods

[0084] Another aspect of the invention pertains to methods of modulatingtarget gene expression, protein expression or activity for therapeuticpurposes. Accordingly, in an exemplary embodiment, the modulatory methodof the invention involves contacting a cell capable of expressing targetgene with a therapeutic agent (e.g., a ss-siRNA or vector or transgeneencoding same) that is specific for the target gene or protein (e.g., isspecific for the mRNA encoded by said gene or specifying the amino acidsequence of said protein) such that expression or one or more of theactivities of target protein is modulated. These modulatory methods canbe performed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a target gene polypeptideor nucleic acid molecule. Inhibition of target gene activity isdesirable in situations in which target gene is abnormally unregulatedand/or in which decreased target gene activity is likely to have abeneficial effect.

[0085] 3. Pharmacogenomics

[0086] The therapeutic agents (e.g., a ss-siRNA or vector or transgeneencoding same) of the invention can be administered to individuals totreat (prophylactically or therapeutically) disorders associated withaberrant or unwanted target gene activity. In conjunction with suchtreatment, pharmacogenomics (i.e., the study of the relationship betweenan individual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a therapeutic agent as wellas tailoring the dosage and/or therapeutic regimen of treatment with atherapeutic agent.

[0087] Pharmacogenomics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, for example, Eichelbaum, M. etal. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder,M. W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0088] One pharmacogenomics approach to identifying genes that predictdrug response, known as “a genome-wide association”, relies primarily ona high-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

[0089] Alternatively, a method termed the “candidate gene approach”, canbe utilized to identify genes that predict drug response. According tothis method, if a gene that encodes a drugs target is known (e.g., atarget gene polypeptide of the present invention), all common variantsof that gene can be fairly easily identified in the population and itcan be determined if having one version of the gene versus another isassociated with a particular drug response.

[0090] As an illustrative embodiment, the activity of drug metabolizingenzymes is a major determinant of both the intensity and duration ofdrug action. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

[0091] Alternatively, a method termed the “gene expression profiling”,can be utilized to identify genes that predict drug response. Forexample, the gene expression of an animal dosed with a therapeutic agentof the present invention can give an indication whether gene pathwaysrelated to toxicity have been turned on.

[0092] Information generated from more than one of the abovepharmacogenomics approaches can be used to determine appropriate dosageand treatment regimens for prophylactic or therapeutic treatment anindividual. This knowledge, when applied to dosing or drug selection,can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with atherapeutic agent, as described herein.

[0093] Therapeutic agents can be tested in an appropriate animal model.For example, an ss-siRNA (or expression vector or transgene encodingsame) as described herein can be used in an animal model to determinethe efficacy, toxicity, or side effects of treatment with said agent.Alternatively, a therapeutic agent can be used in an animal model todetermine the mechanism of action of such an agent. For example, anagent can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

[0094] IV. Pharmaceutical Compositions

[0095] The invention pertains to uses of the above-described agents fortherapeutic treatments as described infra. Accordingly, the modulatorsof the present invention can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the nucleic acid molecule, protein, antibody, or modulatorycompound and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

[0096] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation),transdermal (topical), and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

[0097] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0098] Sterile injectable solutions can be prepared by incorporating theactive compound in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

[0099] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0100] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0101] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0102] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0103] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0104] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0105] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds that exhibit large therapeutic indices arepreferred. Although compounds that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0106] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test compound which achieves a half-maximal response) as determinedin cell culture. Such information can be used to more accuratelydetermine useful doses in humans. Levels in plasma may be measured, forexample, by high performance liquid chromatography.

[0107] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0108] V. Knockout and/or Knockdown Cells or Organisms

[0109] A further preferred use for the ss-siRNA molecules of the presentinvention (or vectors or transgenes encoding same) is a functionalanalysis to be carried out in eukaryotic cells, or eukaryotic non-humanorganisms, preferably mammalian cells or organisms and most preferablyhuman cells, e.g. cell lines such as HeLa or 293 or rodents, e.g. ratsand mice. By administering a suitable ss-siRNA molecules which issufficiently complementary to a target mRNA sequence to directtarget-specific RNA interference, a specific knockout or knockdownphenotype can be obtained in a target cell, e.g. in cell culture or in atarget organism.

[0110] Thus, a further subject matter of the invention is a eukaryoticcell or a eukaryotic non-human organism exhibiting a targetgene-specific knockout or knockdown phenotype comprising a fully or atleast partially deficient expression of at least one endogeneous targetgene wherein said cell or organism is transfected with at least onevector comprising DNA encoding a ss-siRNA molecule capable of inhibitingthe expression of the target gene. It should be noted that the presentinvention allows a target-specific knockout or knockdown of severaldifferent endogeneous genes due to the specificity of the ss-siRNAi.

[0111] Gene-specific knockout or knockdown phenotypes of cells ornon-human organisms, particularly of human cells or non-human mammalsmay be used in analytic to procedures, e.g. in the functional and/orphenotypical analysis of complex physiological processes such asanalysis of gene expression profiles and/or proteomes. Preferably theanalysis is carried out by high throughput methods using oligonucleotidebased chips.

[0112] Using RNAi based knockout or knockdown technologies, theexpression of an endogeneous target gene may be inhibited in a targetcell or a target organism. The endogeneous gene may be complemented byan exogenous target nucleic acid coding for the target protein or avariant or mutated form of the target protein, e.g. a gene or a DNA,which may optionally be fused to a further nucleic acid sequenceencoding a detectable peptide or polypeptide, e.g. an affinity tag,particularly a multiple affinity tag.

[0113] Variants or mutated forms of the target gene differ from theendogeneous target gene in that they encode a gene product which differsfrom the endogeneous gene product on the amino acid level bysubstitutions, insertions and/or deletions of single or multiple aminoacids. The variants or mutated forms may have the same biologicalactivity as the endogeneous target gene. On the other hand, the variantor mutated target gene may also have a biological activity, whichdiffers from the biological activity of the endogeneous target gene,e.g., a partially deleted activity, a completely deleted activity, anenhanced activity etc. The complementation may be accomplished bycompressing the polypeptide encoded by the endogeneous nucleic acid,e.g. a fusion protein comprising the target protein and the affinity tagand the double stranded RNA molecule for knocking out the endogeneousgene in the target cell. This compression may be accomplished by using asuitable expression vector expressing both the polypeptide encoded bythe endogenous nucleic acid, e.g. the tag-modified target protein andthe double stranded RNA molecule or alternatively by using a combinationof expression vectors. Proteins and protein complexes which aresynthesized de novo in the target cell will contain the exogenous geneproduct, e.g., the modified fusion protein. In order to avoidsuppression of the exogenous gene product by the ss-siRNAi molecule, thenucleotide sequence encoding the exogenous nucleic acid may be alteredat the DNA level (with or without causing mutations on the amino acidlevel) in the part of the sequence which so is homologous to thess-siRNA molecule. Alternatively, the endogeneous target gene may becomplemented by corresponding nucleotide sequences from other species,e.g. from mouse.

[0114] VI. Functional Genomics and/or Proteomics

[0115] Preferred applications for the cell or organism of the inventionis the analysis of gene expression profiles and/or proteomes. In anespecially preferred embodiment an analysis of a variant or mutant formof one or several target proteins is carried out, wherein said variantor mutant forms are reintroduced into the cell or organism by anexogenous target nucleic acid as described above. The combination ofknockout of an endogeneous gene and rescue by using mutated, e.gpartially deleted exogenous target has advantages compared to the use ofa knockout cell. Further, this method is particularly suitable foridentifying functional domains of the targeted protein. In a furtherpreferred embodiment a comparison, e.g of gene expression profilesand/or proteomes and/or phenotypic characteristics of at least two cellsor organisms is carried out. These organisms are selected from: (i) acontrol cell or control organism without target gene inhibition, (ii) acell or organism with target gene inhibition and (iii) a cell ororganism with target gene inhibition plus target gene complementation byan exogenous target nucleic acid.

[0116] Furthermore, the RNA knockout complementation method may be usedfor is preparative purposes, e.g. for the affinity purification ofproteins or protein complexes from eukaryotic cells, particularlymammalian cells and more particularly human cells. In this embodiment ofthe invention, the exogenous target nucleic acid preferably codes for atarget protein which is fused to art affinity tag. This method issuitable for functional proteome analysis in mammalian cells,particularly human cells.

[0117] Another utility of the present invention could be a method ofidentifying gene function in an organism comprising the use of ss-siRNAto inhibit the activity of a target gene of previously unknown function.Instead of the time consuming and laborious isolation of mutants bytraditional genetic screening, functional genomics would envisiondetermining the function of uncharacterized genes by employing theinvention to reduce the amount and/or alter the timing of target geneactivity. The invention could be used in determining potential targetsfor pharmaceutics, understanding normal and pathological eventsassociated with development, determining signaling pathways responsiblefor postnatal development/aging, and the like. The increasing speed ofacquiring nucleotide sequence information from genomic and expressedgene sources, including total sequences for the yeast, D. melanogaster,and C. elegans genomes, can be coupled with the invention to determinegene function in an organism (e.g., nematode). The preference ofdifferent organisms to use particular codons, searching sequencedatabases for related gene products, correlating the linkage map ofgenetic traits with the physical map from which the nucleotide sequencesare derived, and artificial intelligence methods may be used to defineputative open reading frames from the nucleotide sequences acquired insuch sequencing projects. A simple assay would be to inhibit geneexpression according to the partial sequence available from an expressedsequence tag (EST). Functional alterations in growth, development,metabolism, disease resistance, or other biological processes would beindicative of the normal role of the EST's gene product.

[0118] The ease with which RNA can be introduced into an intactcell/organism containing the target gene allows the present invention tobe used in high throughput screening (HTS). Solutions containingss-siRNAs that are capable of inhibiting the different expressed genescan be placed into individual wells positioned on a microtiter plate asan ordered array, and intact cells/organisms in each well can be assayedfor any changes or modifications in behavior or development due toinhibition of target gene activity. The amplified RNA can be feddirectly to, injected into, the cell/organism containing the targetgene. Alternatively, the ss-siRNA can be produced from a vector, asdescribed herein. Vectors can be injected into, the cell/organismcontaining the target gene. The function of the target gene can beassayed from the effects it has on the cell/organism when gene activityis inhibited. This screening could be amenable to small subjects thatcan be processed in large number, for example: arabidopsis, bacteria,drosophila, fungi, nematodes, viruses, zebrafish, and tissue culturecells derived from mammals. A nematode or other organism that produces acolorimetric, fluorogenic, or luminescent signal in response to aregulated promoter (e.g., transfected with a reporter gene construct)can be assayed in an HTS format.

[0119] The present invention may be useful in allowing the inhibition ofessential genes. Such genes may be required for cell or organismviability at only particular stages of development or cellularcompartments. The functional equivalent of conditional mutations may beproduced by inhibiting activity of the target gene when or where it isnot required for viability. The invention allows addition of ss-siRNA atspecific times of development and locations in the organism withoutintroducing permanent mutations into the target genome.

[0120] VII. Screening Assays

[0121] The methods of the invention are also suitable for use in methodsto identify and/or characterize potential pharmacological agents, e.g.identifying new pharmacological agents from a collection of testsubstances and/or characterizing mechanisms of action and/or sideeffects of known pharmacological agents.

[0122] Thus, the present invention also relates to a system foridentifying and/or characterizing pharmacological agents acting on atleast one target protein comprising: (a) a eukaryotic cell or aeukaryotic non-human organism capable of expressing at least oneendogeneous target gene coding for said so target protein, (b) at leastone ss-siRNA molecule capable of inhibiting the expression of said atleast one endogeneous target gene, and (c) a test substance or acollection of test substances wherein pharmacological properties of saidtest substance or said collection are to be identified and/orcharacterized. Further, the system as described above preferablycomprises: (d) at least one exogenous target nucleic acid coding for thetarget protein or a variant or mutated form of the target proteinwherein said exogenous target nucleic acid differs from the endogeneoustarget gene on the nucleic acid level such that the expression of theexogenous target nucleic acid is substantially less inhibited by thess-siRNA molecule than the expression of the endogeneous target gene.

[0123] The test compounds of the present invention can be obtained usingany of the numerous approaches in combinatorial library methods known inthe art, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

[0124] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med Chem. 37:1233.

[0125] Libraries of compounds may be presented in solution (e.g.,Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990)Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.)).

[0126] In a preferred embodiment, the library is a natural productlibrary, e.g., a library produced by a bacterial, fungal, or yeastculture. In another preferred embodiment, the library is a syntheticcompound library.

[0127] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein by reference.

EXAMPLES Overview of Examples I-II

[0128] How siRNAs direct target cleavage and whether a single mechanismexplains the function of siRNAs in post-transcriptional gene silencingin plants, quelling in fungi, and RNAi in animals remain unknown.Furthermore, how siRNAs are permitted to enter the RNAi pathway whileother 21-23 nt RNAs seem to be excluded cannot yet be fully explained.

[0129] Three models have been proposed for RNAi in Drosophila. Eachmodel seeks to explain the mechanism by which siRNAs direct target RNAdestruction. In one model target destruction requires an RNA-dependentRNA polymerase (RdRP) to convert the target mRNA into dsRNA (Lipardi etal. (2001) Cell 107:297-307). The RdRP is hypothesized to usesingle-stranded siRNAs as primers for the target RNA-templated synthesisof complementary RNA (cRNA). The resulting cRNA/target RNA hybrid isproposed to then be cleaved by Dicer, destroying the mRNA and generatingnew siRNAs in the process. Key features of this model are that theATP-dependent, dsRNA-specific endonuclease Dicer acts twice in the RNAipathway, that target destruction should require nucleotide triphosphatesto support the production of cRNA, and that a 3′ hydroxyl group isessential for siRNA function, since siRNAs are proposed to serve asprimers for new RNA synthesis.

[0130] A second model proposes that single-stranded siRNAs do not act asprimers for an RdRP, but instead assemble along the length of the targetRNA and are then ligated together by an RNA ligase to generate cRNA(Lipardi et al. (supra); Nishikura (2001) Cell 107:415-418). ThecRNA/target RNA hybrid would then be destroyed by Dicer. This modelpredicts that target recognition and destruction should require ATP (orperhaps an NAD-derived high energy cofactor) to catalyze ligation, aswell as ATP to support Dicer cleavage. Like the first model, theligation hypothesis predicts that an siRNA 3′ hydroxyl group will beabsolutely required for RNAi. Furthermore, a 5′ phosphate should berequired for siRNA ligation. Unlike the first model, however,ribonucleotide triphosphates other than ATP should not be required fortarget destruction.

[0131] A third model (FIG. 1) hypothesizes that two distinct enzymes orenzyme complexes act in the RNAi pathway (Hammond et al. (2000) Nature404:293-296; Zamore et al. (supra); and Nykänen et al. (supra)). As inthe first model, Dicer is proposed to generate siRNAs from dsRNA. ThesesiRNAs are then incorporated into a second enzyme complex, theRNA-induced silencing complex (RISC), in an ATP-dependent step or seriesof steps during which the siRNA duplex is unwound into single strands.The resulting single-stranded siRNA is proposed to guide the RISC torecognize and cleave the target RNA in a step or series of stepsrequiring no nucleotide cofactors whatsoever. The absence of anucleotide triphosphate requirement for target recognition and cleavageis a key feature of this model.

[0132] The present inventors have previously demonstrated by twodifferent experimental protocols that both recognition andendonucleolytic cleavage of a target RNA proceeds efficiently in thepresence of less than 50 nM ATP, a concentration presumed to beinsufficient to support either the synthesis of new RNA or the ligationof multiple siRNAs into cRNA (Nykänen et al. (supra)). However, thesedata also revealed an absolute requirement for a 5′ phosphate for siRNAsto direct RNAi in Drosophila embryo lysates, a finding that wasinterpreted to reflect an authentication step in the assembly of theRNAi-enzyme complex, the RISC. It was envisioned that the 5′ phosphatewas involved in obligatory non-covalent interactions with one or moreprotein components of the RNAi pathway. Nonetheless, the 5′ phosphaterequirement might formally reflect a requirement for the phosphate groupin covalent interactions, such as the ligation of multiple siRNAs togenerate cRNA (Nishikura (2001) Cell 107:415-418).

[0133] To distinguish among these three models, the requirement for a 5′phosphate and a 3′ hydroxyl group on the anti-sense strand of the siRNAduplex was examined. First, the role of these functional groups in RNAiusing both Drosophila embryo lysates and human HeLa S100 extract wasexamined. Human HeLa S100 extract, like Drosophila embryo lysates,supports RNAi in vitro. Second, the findings were validated in vivo inhuman HeLa cells. The data support a model for the RNAi pathway in whichsiRNAs function as guides for an endonuclease complex that mediatestarget RNA destruction. The data demonstrate that the requirement for a5′ phosphate is conserved between Drosophila and human cells, but thatin neither organism is an siRNA 3′ hydroxyl needed. There was noevidence that RdRPs play any role whatsoever in Drosophila or humanRNAi, despite the clear requirement for such enzymes in PTGS in plants,quelling in Neurospora crassa, and RNAi in C. elegans and Dictyosteliumdiscoideum (Cogoni et al. (1999) Nature 399:166-169; Dalmayetal. (2000)Cell 101:543-553; Mourrain et al. (2000) Cell 101, 533-542; Smardon etal. (2000) Curr. Biol. 10:169-178; Sijen et al. (2001) Cell 107:465-476;Martens et al. (supra)). In this respect, the mechanism of RNAi in fliesand mammals is distinct from that of PTGS, quelling, and RNAi in wormsand Dictyostelium.

Example I Requirement for the siRNA 5′ Phosphate in Human RNAi

[0134] Synthetic siRNAs bearing a 5′ hydroxyl can efficiently mediateRNAi both in vitro in Drosophila embryo lysates and in vivo in culturedhuman cells (Elbashir et al. (2000b supra); Elbashir et al. (2000bsupra); and Nykänen et al. (supra)). However, in the Drosophila in vitrosystem, an endogenous kinase rapidly converts the 5′ hydroxyl group to aphosphate (Nykänen et al. (supra)). Blocking siRNA phosphorylation bysubstituting the 5′ hydroxyl with a methoxy moiety completely blocksRNAi in Drosophila embryo lysates (Nykänen et al. (supra)). Furthermore,5′ phosphorylated siRNAs more efficiently trigger RNAi in vivo inDrosophila embryos than do 5′ hydroxyl-containing siRNAs (Boutla et al.(2001) Curr. Biol. 11 :1776-1780). 5′ hydroxyl-containing, syntheticsiRNAs that trigger RNAi in cultured mammalian cells (Elbashir et al.(2001a supra); Elbashir et al. (2002 supra)), in mice (McCaffrey et al.(2002) Nature 418:38-39; Lewis et al. (2002) Nat. Genet. 10.1038/ng.944)and perhaps even in plants (Klahre et al. (2002) 14 Aug. 2002(10.10731pnas. 182204199) may likewise be phosphorylated by a cellularkinase prior to entering the RNAi pathway.

[0135] To determine if a 5′ phosphate is required for RNAi in mammals,mammalian RNAi was first examined in vitro, using HeLa cell S100extract. These reactions accurately recapitulate the known features ofsiRNA-directed RNAi in mammalian cell culture: exquisitesequence-specificity (Elbashir et al. (2001a supra)) and target RNAcleavage (Holen et al. (2002) Nucleic Acids Res. 30:1757-1766). RNAireactions were performed in HeLa S100 extracts using siRNA duplexes inwhich the guide strand (i.e., the antisense strand) contained either a5′ hydroxyl or a 5′ methoxy group (FIG. 2A) and a chimeric target RNA inwhich nucleotides 62 to 81 were complementary to the siRNA (FIG. 2B).When the guide strand of the siRNA duplex contained a 5′ hydroxyl group,and could, therefore, be phosphorylated, it directed cleavage of thetarget RNA within the sequence complementary to the siRNA (FIG. 3).Target cleavage directed by this siRNA occurred at precisely the samesite in the HeLa S100 as in Drosophila embryo lysate. These data suggestthat endonucleolytic cleavage of the target RNA is a common feature ofRNAi in flies and mammals. siRNAs with a 5′ methoxy group cannot bephosphorylated by nucleic acid kinases and cannot direct RNAi in lysatesof Drosophila embryos (Nykänen et al. (2001 supra)). Such siRNAs werelikewise unable to direct cleavage of the target RNA in the HeLa S100reaction (FIG. 3A). Although the exogenous, methoxy-blocked siRNA doesnot trigger RNAi in these reactions, an endogenous HeLa RISC complexthat contains the miRNA, let-7 (Hutvágner and Zamore (2002) 1 Aug. 2002(10.1126/science.1073827, cleaved the chimeric target RNA within thelet-7 complementary sequence near its 3′ end (FIG. 2B) in all of thehuman invitro RNAi reactions. This 5′ cleavage product (indicated by anasterisk) serves as an internal control for the HeLa reactions. Thus,mammalian RNAi, like RNAi in Drosophila (Boutla et al. (2001) Curr.Biol. 11:1776-1780; Nykänen et al. (2001 supra)), requires the siRNA 5′phosphate for target cleavage and suggest that 5′ hydroxyl-containingsiRNA duplexes must be phosphorylated by a cellular kinase before theybecome competent to mediate RNAi in human cells. Consistent with thisidea, 5′ hydroxyl-containing siRNAs are rapidly 5′ phosphorylated afteronly 5 min incubation in the HeLa S100 (FIG. 3B). Thus, like Drosophila,human cells contain a nucleic acid kinase that can add a 5′ phosphate toa synthetic siRNA.

Example II Role of the siRNA 3 Hydroxyl Group in Flies and Mammals

[0136] Both siRNAs produced by enzymatic cleavage of dsRNA and thoseprepared by chemical synthesis contain 3′ hydroxyl termini (Elbashir etal. (supra)). Experiments using nuclease-treated siRNAs suggested that a3′ phosphate blocks RNAi in Drosophila embryo lysates (Lipardi et al.(supra)), a finding consistent with authentication of siRNA 3′ structureby the RNAi machinery, with siRNAs acting as primers for cRNA synthesis,or with RNA-templated ligation of multiple siRNAs into cRNA. Todetermine if the siRNA 3′ hydroxyl group plays an essential role inRNAi, two siRNAs were synthesized in which the 3′ hydroxyl group of theguide strand was blocked (FIG. 2). In one siRNA, the 3′ hydroxyl wasreplaced by a 2′,3′ dideoxy terminus. In the other, the 3 positioncontained 3-amino-propyl phosphoester (3′ ‘amino modifier’). Each of theblocked siRNA guide strands was analyzed by electrospray massspectrometry to confirm its identity and purity. The two modified siRNAguide strands, as well as a 3′ hydroxyl-containing control strand, wereannealed to a standard 21 nt siRNA sense strand. The three resultingsiRNA duplexes were tested for their ability to direct cleavage of acomplementary target RNA in an in vitro RNAi reaction containingDrosophila embryo lysate. FIG. 4A shows that the two 3′-blocked siRNAsproduced the same degree of target cleavage as the 3′hydroxyl-containing siRNA control.

[0137] Next, the experiment was repeated in HeLa S100 extract todetermine if an siRNA 3′ hydroxyl group is required for RNAi inmammalian cells. 3′ modification of an siRNA has been reported to bepermitted for RNAi in mammalian cells (Holen et al. (supra)), but it wasnot shown in those experiments that all of the siRNA was 3′ modified. Incontrast to the 5′ methoxy modification, which completely blocked targetRNA cleavage in the HeLa S100 reaction, 3′ modification had no effect onthe efficiency or specificity of RNAi (FIG. 4B). The identity and purityof these siRNAs was confirmed by electrospray mass spectrometry.However, it could be envisioned that a fraction of the siRNA guidestrand was cleaved within the single-stranded, two nucleotide, 3′overhang by a nuclease in the HeLa S100, regenerating the 3′ hydroxyl.If this occurred, the cleaved siRNAs could then act as primers. Toexclude this possibility, RNAi reactions were performed usingprogressively shorter guide siRNAs blocked at the 3′ end by either a2′,3′ dideoxy or a 3′ amino modifier group. The 20 or 19 nt guidestrands were annealed to the same 21 nt sense siRNA strand. FIG. 4Bshows that target RNA cleavage occurred in all cases, although theefficiency of cleavage decreased as the siRNA guide strand wasshortened, even when it contained a 3′ hydroxyl terminus. If the 3′blocked 21 nt siRNA was active because it had been shortened to a20-mer, it could not have attained the activity of the 3′ hydroxyl 21 ntsiRNA. Similarly, if nucleolytic removal of the 3′ block accounted forthe activity of the 20 nt guide siRNA, it should have only been asactive as the 19 nt, 3′ hydroxyl-containing siRNA. These results suggestthat the 3′ hydroxyl group of the siRNA guide strand does not play anobligatory role in siRNA-directed RNAi in flies or mammals.

Example III Single-stranded siRNAs

[0138] Current models for RNAi, including those that propose siRNA tofunction as guides for an endonuclease and models that propose siRNAs toact as primers for target-RNA templated RNA synthesis, predict thatsiRNAs ultimately function as single strands. In fact, in Drosophilaembryos, single-stranded antisense siRNAs corresponding to the NotchmRNA elicited Notch phenotypes in 12% of injected embryos, although theexpressivity was quite low (Boutla et al. (2001, supra)). Furthermore,single-stranded RNAs of various lengths trigger RNAi in C. elegans, butonly when they contain a 3′ hydroxyl group, suggesting thatsingle-stranded siRNA functions in that organism as a primer for an RdRP(Tijsterman et al. (2002, supra). Consistent with single-stranded siRNAsacting in nematodes as primers that direct the production of new dsRNA,they fail to trigger RNAi in the absence of Dicer (Dcr-1) (Tijsterman etal. (2002, supra)).

[0139] It was next examined if the guide siRNA strand alone couldtrigger target cleavage in an in vitro RNAi reaction containing eitherDrosophila embryo lysate or human HeLa cell S100. First, it was examinedwhether single-stranded siRNA could direct target RNA cleavage inDrosophila embryo lysates (FIG. 5A). For this experiment, siRNA was usedhaving the sequence of the miRNA let-7 (FIG. 2A). Cleavage of the targetRNA (FIG. 2B) by a let-7-containing siRNA duplex produces a diagnostic522 nt 5′ product (Hutvágner and Zamore, 2002a). When the syntheticsiRNA was used as a single strand, the target RNA was not cleaved (FIG.5A). Similarly, a single-stranded siRNA of the same sequence but bearinga 2′ deoxy thymidine (dT) instead of uracil as its first nucleotide, wasalso a poor trigger of target cleavage. However, both these siRNAscontain a 5′ hydroxyl, and a 5′ phosphate is required for siRNA duplexesto trigger target RNA cleavage in Drosophila embryo lysates (Nykänen etal., 2001). It was thus hypothesized that the defect with thesingle-stranded siRNAs might be that they lacked a 5′ phosphate andcannot obtain one because they are not substrates for the Drosophilakinase. In support of this hypothesis, when the single-stranded siRNAstarting with dT was pre-phosphorylated with polynucleotide kinase, itdirected target cleavage.

[0140] To confirm these findings, the activity of a secondsingle-stranded siRNA, complementary to the luciferase portion of thetarget RNA, was examined. When pre-phosphorylated, this single-strandedsiRNA again directed target cleavage in Drosophila embryo lysate, albeitless efficiently than the same molar concentration of an siRNA duplex(FIG. 5B). Cleavage occurred at precisely the same site in the targetRNA for both single-stranded and double-stranded siRNAs, suggesting thatthe single-stranded siRNA entered the RNAi pathway, rather thantriggered RNA destruction by a different route. The same single-strandedsiRNA sequence bearing a 5′ methoxy group did not direct target RNAcleavage (FIG. 5B). Together, the experiments in FIG. 5 demonstrate thatsingle-stranded siRNAs, like the guide strands of siRNA duplexes, do notfunction in the RNAi pathway unless they bear a 5′ phosphate.

[0141] To determine if single-stranded siRNAs trigger target destructionin Drosophila embryo lysates by acting as primers, the 3′ end of thesiRNA to 2′,3′ dideoxy was modified. As with double-stranded siRNAs,blocking the 3′ end of the single-stranded siRNA had no effect on theefficiency or specificity with which the target was cleaved (FIG. 5B).It should be noted that the efficiency of target cleavage bysingle-stranded siRNAs is significantly less than that of siRNAduplexes. The lower efficiency might simply reflect the remarkably shortlifespan of single-stranded siRNA in the Drosophila embryo lysate: thevast majority is destroyed within the first 2 min of incubation. Oneexplanation for the requirement for a 5′ phosphate might be that withoutit, the single-stranded siRNA is destroyed even faster. This explanationis unlikely, because the rate of single-stranded RNA destruction is only1.4-fold faster for 5′ hydroxy siRNAs (FIG. 5C). More likely is that the5′ phosphate of the single-stranded siRNA is required for its entry intothe RISC, and that because a small fraction of 5′ phosphorylated,single-stranded siRNA enters the RISC it is protected from degradation,enhancing its stability in the lysate.

[0142] Next, it was examined whether single-stranded siRNAs couldfunction to trigger RNAi in HeLa S100 extracts. Again, single-strandedsiRNAs directed target cleavage at the same site as the correspondingsiRNA duplex (FIG. 6A). Pre-phosphorylation of single-stranded siRNA wasnot required for it to function in target cleavage in HeLa S100, butblocking the 5′ end with a methoxy group completely eliminated RNAi(FIG. 6B). These results suggest that a 5′ phosphate is required formammalian RNAi, but that the nucleic acid kinase(s) responsible forphosphorylating siRNAs in HeLa S100 acts on single-stranded siRNA,unlike its Drosophila counterpart. Blocking the 3′ end of thesingle-stranded siRNA had no effect on the ability of thesingle-stranded siRNA to cleave the target RNA in HeLa S100 (FIG. 6A).Thus, the structural requirements for single-stranded siRNA function intarget cleavage are conserved between flies and mammals: a 5′ phosphateis required, but a 3′ hydroxyl is not.

[0143] Together these data support the view that siRNAs do not directtarget RNA destruction by priming the synthesis of new RNA, nor aresiRNAs ligated together to generate cRNA. Both processes should requirea 3′ hydroxyl group, which is dispensable for target cleavage in eitherDrosophila or human cell extracts. Instead, the data suggest that siRNAsact as guides to direct a protein endoribonuclease to cleave the targetRNA. The finding that single-stranded siRNAs can function as guides inthe RNAi pathway suggests that each individual RISC contains only onesiRNA strand. Consistent with this view, in HeLa cell S100 extracts, thesingle stranded miRNA, let-7, is in an endogenous RISC that catalyzesmultiple rounds of cleavage of a perfectly complementary target RNA(Hutvágner and Zamore, 2002a).

[0144] Previously, it was proposed that the siRNA 5′ phosphate wasrecognized twice during the assembly of the siRNA-containingendoribonuclease complex (Nykänen et al., 2001). That study placed one5′ phosphate recognition event before siRNA duplex unwinding, but couldnot distinguish whether the 5′ phosphate is required subsequently at theunwinding step itself or after unwinding is complete. The absence oftarget cleavage by single-stranded siRNAs lacking a 5′ phosphatesuggests that the second phosphate recognition step occurs after thesiRNA duplex is unwound. In both Drosophila embryo lysates and humanHeLa S100, cleavage directed by single-stranded siRNA was less efficientthan RNAi triggered by siRNA duplexes. This inefficiency correlated withthe general instability of short RNA in the in vitro extracts, asdetermined by measuring single-stranded siRNA half-life using 3′radiolabeled siRNAs (FIG. 5C) and by Northern hybridization.

Example IV siRNAs Do Not Function as Primers in HeLa Cells

[0145] To assess if the above-described in vitro results accuratelypredict the RNAi mechanism in vivo, cultured human cells were used toassess the structural requirements for siRNA function. Synthetic siRNAswere co-transfected into HeLa cells with plasmids expressing target(Photinus pyralis, Pp) and control (Renilla reniformis, Rr) luciferasemRNAs. Luciferase expression was measured, and target (firefly)luciferase levels were normalized to the Renilla control. The results ofthese experiments are shown in FIG. 7.

[0146] First, the requirement for a 5′ phosphate observed in Drosophilaand HeLa extracts was conserved in vivo (FIG. 7A). A 5′hydroxyl-containing siRNA duplex triggered efficient gene silencing invivo, reducing expression of the target luciferase >90%. In contrast, a5′ methoxy-modified siRNA reduced firefly luciferase levels by onlytwo-fold. This small reduction may reflect inhibition of translation,perhaps by an anti-sense mechanism. Alternatively, some of themethoxy-blocked siRNA may inefficiently enter the RNAi pathway in vivo.An siRNA in which the guide strand contained a 5′ amino modifier group,6-amino-hexyl phosphoester, was significantly more effective insuppressing target mRNA expression than the siRNA with the 5′ methoxygroup (FIG. 7A). This finding is consistent with the idea that a 5′phosphate group is required for siRNA function, but that the 5′phosphate participates in non-covalent interactions only, since themodified 5′ phosphate should be less able to act as an electronacceptor. The in vivo studies agree with the in vitro results: a 5′phosphate is essential for efficient siRNA function in flies andmammals. However, in flies only duplex siRNAs can be 5′ phosphorylatedby cellular kinases, whereas in mammals, both single-stranded anddouble-stranded siRNAs are phosphorylated.

[0147] Consistent with the view that the core function of siRNA in humancells is as guides, not primers, blocking the 3′ end of the siRNA guidestrand had no effect on RNAi in vivo. siRNA duplexes in which the guidestrand contained a 3′ hydroxyl, a 2′,3′ dideoxy, or a 3′ amino modifierwere all equally effective in triggering RNAi in vivo (FIG. 7A). Thesilencing activity in vivo of a 21-nt, 3′-blocked siRNA guide strand wasgreater than that of a 20-nt, 3′ hydroxy siRNA guide strand, indicatingthat the 3′ block was not removed in vivo. It is believed that thesedata exclude an obligatory role for the siRNA 3′ hydroxyl group in RNAiin mammalian cells, and argue that siRNAs do not normally trigger targetdestruction in human cells by functioning as primers.

[0148] These experiments were conducted at siRNA concentrations wherethe siRNA is not limiting for RNA silencing. An siRNA function inpriming the synthesis of dsRNA might be used when siRNAs are limiting.Therefore, the relative ability of siRNA duplexes in which the guidestrand either contained a 3′ hydroxyl or a 2′,3′ dideoxy group at lowsiRNA concentrations was tested (FIG. 7B). It was found that theefficacy of the two types of siRNAs did differ when siRNA was ratelimiting for target mRNA silencing, but never by more than 1.8-fold. Theobserved difference in efficacy between the two types of siRNAs is notsufficiently great to support the view that the 3′ hydroxyl group of thesiRNA is used to prime the synthesis of dsRNA from the target mRNA. Ifthe siRNA were used to prime dsRNA synthesis, the production of newdsRNA by an RdRP using the siRNA as a primer should have amplified thesilencing activity of the 3′ hydroxy but not the 2′,3′ dideoxy siRNA atlimiting concentrations. To further exemplify, if the 3′ hydroxy guidestrand had primed synthesis of one molecule of dsRNA (˜130 bp long basedon the site of siRNA/target complementarity) for each target mRNAmolecule, and this new dsRNA was then “Diced” into just two of thepossible six new siRNAs, at least a two-fold difference between the twosiRNAs should have been observed. This analysis even further fails toaccount for the new crop of siRNAs acting in a subsequent cycle ofpriming, which would further amplify the difference between 3′ deoxy and3′ hydroxy siRNA at limiting concentrations. The simplest interpretationof the above findings that 3′ hydroxy siRNAs trigger no significantamplification of RNA silencing relative to 3′ blocked siRNAs is that ansiRNA-primed, RdRP-dependent cycle of siRNA amplification plays noproductive role in RNAi in cultured HeLa cells, even at low siRNAconcentrations. The small difference in efficacy between 3′ OH and 2′,3′dideoxy siRNAs likely indicates that the blocked siRNAs have a subtledefect such as a lower affinity for components of the RNAi machinery,slightly reduced intracellular half-life, or a minor reduction inphosphorylation rate. This defect may result from the 2′ deoxymodification of the terminal nucleotide, rather than the 3′ block, sincesiRNAs with 2′ deoxythymidine tails have been reported to be lessefficient than those containing uracil in HeLa cells (Hohjoh (2002) FEBSLetts. 521:195-199).

[0149] The above in vitro studies indicate that single-stranded siRNAscan enter the RNAi pathway, albeit inefficiently. To test ifsingle-stranded siRNAs could trigger mRNA silencing in vivo, variousconcentrations of single-stranded, sense or antisense siRNA weresubstituted for siRNA duplexes in HeLa cell co-transfections (FIG. 8A).As the concentration of antisense single strand was increased, theexpression of the firefly luciferase decreased relative to the Renillainternal control. Single-stranded siRNAs were less efficient than siRNAduplexes: it took nearly 8-times more single-stranded siRNA to approachthe potency of the corresponding duplex. This inefficiency may simplyreflect rapid degradation of the majority of the transfectedsingle-stranded siRNA before it can enter the RISC complex. Cells maypossess a mechanism that stabilizes siRNA duplexes and shuttles them tothe RISC as single-strands without exposing them to degradatory enzymes.Thus, if endogenous siRNAs are double-stranded in vivo, they may bedouble-stranded so as to facilitate their entry into the RNAi pathwayand to exclude them from a competing pathway that degrades small,single-stranded RNA. Alternatively, single-stranded siRNAs may bypass akey step in RISC assembly, making them less efficient than duplexes intriggering RNAi. The dramatic instability of single-stranded siRNAs invitro may simply reflect their inefficiency in assembling into a RISC,which could protect them from degradation.

[0150] Gene silencing by single-stranded siRNA was sequence-specific,and single-stranded sense siRNA did not alter the expression of thetarget RNA (FIG. 8B). Thus, it is unlikely that siRNAs themselves arecopied by an RdRP in mammalian cells, since copying the sense siRNAshould generate the anti-sense siRNA strand. However, copying sensesiRNA into a duplex would not generate the characteristic 3′ overhangingends of siRNAs. Such 3′ overhangs might be required for siRNA unwindingand/or efficient RISC assembly. Pre-phosphorylation of single-strandedsiRNA did not enhance its potency in HeLa cells, consistent with theobservations in HeLa S100 extracts, but blocking phosphorylation with a5′ methoxy group abolished silencing, pointing to the importance of 5′phosphorylation for single-stranded siRNA function in vivo (FIG. 8B).The exemplified findings are not entirely unexpected, since endogenous,single-stranded miRNAs enter the RNAi pathway in HeLa cells (Hutvágnerand Zamore, (2002a supra). In particular, the finding thatsingle-stranded siRNAs can elicit RNA silencing blurs the distinctionbetween RNAi and antisense effects. The above data evidence thatsingle-stranded siRNAs trigger the same pathway as siRNA duplexes: bothguide endonucleolytic cleavage of target RNAs at the same site, and bothrequire 5′ phosphates, but not 3′ hydroxyl groups, to function. The datasupport the view that single-stranded siRNAs function in the samepathway as siRNA duplexes, the RNAi pathway.

[0151] The in vitro experiments with Drosophila embryo lysates and HeLaS100 extracts and in vivo experiments in HeLa cells argue against siRNAsfunctioning as primers in the RNAi pathway. These findings areconsistent with the absence of any genes encoding canonical RdRPs in thecurrently available release of either the Drosophila or human genome. Ahallmark of the involvement of RdRPs in post-transcriptional silencingis the spread of silencing beyond the confines of an initial triggerdsRNA or siRNA into regions of the target RNA 5′ to the silencingtrigger. In C. elegans, this spreading (‘transitive RNAi’) is manifestin the production of new siRNAs corresponding to target sequences notcontained in the exogenous trigger dsRNA (Sijen et al. (2001, supra)).Furthermore, small RNAs as long as 40 nt can initiate silencing inworms, but only if they contain 3′ hydroxyls, suggesting that they actas primers for the synthesis of cRNA (Tijsterman et al. (2002, supra).In contrast, 5′ spreading is not detected in Drosophila, either in vitro(Zamore et al. (2000, supra)), in cultured Drosophila S2 cells (Celottoand Graveley, (2002, supra)), or in vivo in flies. The data support theview that, in both flies and mammals, siRNAs trigger target RNAdestruction not by acting as primers, but rather by guiding a proteinendoribonuclease to a site on the target RNA that is complementary toone strand of the siRNA. The observation that the target cleavage siteis across from the center of the complementary siRNA (Elbashir et al.(2001c, supra); Elbashir et al. (2001b, supra)) is consistent with anenzyme other than Dicer acting in target RNA destruction and not withmodels that propose that Dicer destroys target RNAs. Furthermore,mammalian extracts depleted of Dicer still catalyze siRNA-directedtarget cleavage (Martinez et al. (2002, in press).

[0152] Previous concerns have been raised regarding the possibility ofdesigning siRNAs capable of degrading a particular mRNA isoform thatdiffers from other isoforms in only a small region of sequence, perhapsa single nucleotide. If siRNAs do not act as RdRP primers in flies andmammals, then there is no fear that the silencing signal will spread 5′to a region of sequence common to the entire family of mRNAs. Thus,despite earlier concerns that such siRNAs would not be possible(Nishikura, 2001), these data suggest that isoform- andpolymorphism-specific siRNAs can be designed for use in mammals todissect the function of individual gene isoforms and also fortherapeutic use to treat, for example, inherited autosomal dominanthuman diseases.

[0153] The above examples demonstrate that the anti-sense strand of ansiRNA can function in RNAi in vitro in Drosophila or human cell extractsin the absence of the sense siRNA strand. These single-stranded siRNAsonly function if they are 5′ phosphorylated. 5′ phosphorylated,single-stranded siRNA directed target RNA cleavage in Drosophila embryolysates, albeit less efficiently than the same molar concentration ofthe corresponding siRNA duplex. Cleavage of a target RNA in response toa 5′ phosphorylated, single-stranded siRNA occurred at precisely thesame site in the target as was observed for a standard siRNA duplex.Thus, single-stranded siRNA enters the RNAi pathway. The 2′ and 3′positions of the siRNA terminus are amenable to chemical modification,since modifying the 3′ end of the siRNA to 2′,3′ dideoxy does not impairits function. Thus, it is now possible to introduce specific chemicalmodifications into the single-stranded siRNA to enhance its in vivostability. The requirement for a 5′ phosphate is absolute, sinceblocking 5′ phosphorylation of the single-stranded siRNA by introducinga 5′ methoxy group completely eliminated its ability to direct targetRNA cleavage. Modification of the 5′ most nucleotide of an siRNAantisense strand from ribo to 2′ deoxy was previously shown to inhibitphosphorylation of the siRNA in Drosophila embryo lysates. However,pre-phosphorylating the 5′ end of this 2′ deoxy-containing siRNA allowsit to function as a single-strand. Thus, 5′ modifications of thesingle-stranded siRNA are also possible, as long as the single-strandedsiRNA is 5′ phosphorylated.

[0154] In both Drosophila embryo lysates and human HeLa S100, cleavagedirected by single-stranded siRNA was less efficient than RNAi triggeredby siRNA duplexes. This inefficiency correlated with the generalinstability of short RNA in the in vitro extracts, as determined bymeasuring single-stranded siRNA half-life using 3′ radiolabeled siRNAsand by using Northern hybridization. If siRNAs siRNAs aredouble-stranded in vivo, they may be double-stranded so as to facilitatetheir entry into the RNAi pathway and to exclude them from a competingpathway that degrades small, single-stranded RNA. Thus, a preferredaspect of the invention features single-stranded siRNAs which have beenmodified so as to enhance their in vivo stability, while still retainingtheir function in triggering sequence-specific RNAi.

[0155] Key improvements over the prior art include, but are not limitedto, the following: (1) single-stranded siRNAs are smaller, lesscomplicated and less expensive to produce than siRNA duplexes; and (2)chemically-modified anti-sense RNA oligonucleotides (single-strandedsiRNAs) trigger RNA interference in an efficient, naturally-occurringcellular pathway for the targeted destruction of an mRNA. Chemicalmodification of single-stranded siRNA is compatible with its functionand enhances siRNA stability. The single-stranded siRNAs of the instantinvention are particularly useful in, for example, functional genomicsand drug target validation. Moreover, the modified single-strandedsiRNAs of the instant invention are useful therapeutically in targetinggenes of interest in vivo for RNA interference.

[0156] Experimental Procedures

[0157] General Methods

[0158]Drosophila embryo lysate preparation, in vitro RNAi reactions, andcap-labeling of target RNAs using Guanylyl transferase were carried outas previously described (Zamore et al. (supra)). Human S100 extractswere prepared as described (Dignam et al. (1983) Nucleic Acids Res.11:1475-1489). HeLa S100 was substituted for Drosophila embryo lysate inan otherwise standard RNAi reaction, except that incubation was at 37°C. instead of 25° C. Cleavage products of RNAi reactions were analyzedby electrophoresis on 8% denaturing acrylamide gels. Gels were dried,exposed to image plates (Fuji), which were scanned with a Fuji FLA-5000phosphorimager. Images were analyzed using Image Reader FLA-5000 version1.0 (Fuji) and Image Gauge version 3.45 (Fuji).

[0159] siRNA Preparation

[0160] Synthetic RNAs (Dharmacon) were deprotected according to themanufacturer's protocol and processed as previously described (Nykänenet al. (supra)). siRNA strands were annealed (Elbashir et al. (supra))and used at 100 nM final concentration unless otherwise noted. siRNAsingle strands were phosphorylated with polynucleotide kinase (NewEngland Biolabs) and 1 mM ATP according to the manufacturer'sdirections.

[0161] Tissue Culture

[0162] siRNA transfections were as described (Elbashir et al. (supra)).Briefly, HeLa cultured cells were propagated in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and1% penicillin/streptomycin (Life Technologies). Cells were trypsinizedand seeded at 1×10 5 cells/ml in 24 well plates (5×10 4 cells/well).Twenty four hours after seeding, 1 mg pGL2 control firefly luciferase(Pp-luc GL2; Promega) and 0.1 mg pRL-TK Renilla luciferase (Rr-luc;Promega) plasmids and the luciferase siRNA (25 nM) were co-transfectedwith LipofectAMINE 2000 reagent (Invitrogen) in DMEM (Life Technologies)lacking serum and antibiotics according to manufacturer's instructions.Media was replaced 4 h after transfection with DMEM containing 10% fetalbovine serum (Life Technologies), and cells were lysed 2 days aftertransfections in 1× Passive Lysis Buffer (Promega) according to themanufacturer's instructions. Luciferase expression was determined by theDual luciferase assay kit (Promega) using a Mediators PhL luminometer.Data analysis was performed using Excel (Microsoft) and IgorPro 5.0(Wavemetrics). All experiments were performed in triplicate, and errorwas propagated through all calculations.

[0163] Equivalents

[0164] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 24 1 21 RNA Artificial Sequence synthetic 1 ucgaaguauu ccgcguacgu g 212 21 RNA Artificial Sequence synthetic 2 cguacgcgga auacuucgau u 21 3 21DNA Artificial Sequence synthetic combined DNA/RNA sequence 3 ncgaaguauuccgcguacgu g 21 4 21 RNA Artificial Sequence synthetic 4 cguacgcggaauacuucgau u 21 5 21 RNA Artificial Sequence synthetic 5 ucgaaguauuccgcguacgu n 21 6 21 RNA Artificial Sequence synthetic 6 cguacgcggaauacuucgau u 21 7 21 RNA Artificial Sequence synthetic 7 ucgaaguauuccgcguacgu n 21 8 21 RNA Artificial Sequence synthetic 8 cguacgcggaauacuucgau u 21 9 20 RNA Artificial Sequence synthetic 9 ucgaaguauuccgcguacgu 20 10 21 RNA Artificial Sequence synthetic 10 cguacgcggaauacuucgau u 21 11 20 RNA Artificial Sequence synthetic 11 ucgaaguauuccgcguacgn 20 12 21 RNA Artificial Sequence synthetic 12 cguacgcggaauacuucgau u 21 13 20 RNA Artificial Sequence synthetic 13 ucgaaguauuccgcguacgn 20 14 21 RNA Artificial Sequence synthetic 14 cguacgcggaauacuucgau u 21 15 20 RNA Artificial Sequence synthetic 15 ucgaaguauuccgcguacgc 20 16 21 RNA Artificial Sequence synthetic 16 cguacgcggaauacuucgau u 21 17 19 RNA Artificial Sequence synthetic 17 ucgaaguauuccgcguacg 19 18 21 RNA Artificial Sequence synthetic 18 cguacgcggaauacuucgau u 21 19 19 RNA Artificial Sequence synthetic 19 ucgaaguauuccgcguacn 19 20 21 RNA Artificial Sequence synthetic 20 cguacgcggaauacuucgau u 21 21 21 RNA Artificial Sequence synthetic 21 ugagguaguagguuguauag u 21 22 21 RNA Artificial Sequence synthetic 22 uauacaaccuacuaccucau u 21 23 21 RNA Artificial Sequence synthetic 23 ngagguaguagguuguauag n 21 24 21 RNA Artificial Sequence synthetic 24 uauacaaccuacuaccucau u 21

What is claimed:
 1. An isolated, single-stranded small interferingmolecule (ss-siRNA), wherein the sequence of said ss-siRNA molecule issufficiently complementary to a target mRNA sequence to directtarget-specific RNA interference (RNAi) and wherein the 5′ nucleotide is5′ phosphorylated or is capable of being 5′ phosphorylated in situ or invivo.
 2. The ss-siRNA of claim 1 which is sufficiently complementary toa target mRNA, said target mRNA specifying the amino acid sequence of acellular protein.
 3. The ss-siRNA of claim 1 which is sufficientlycomplementary to a target mRNA, said target mRNA specifying the aminoacid sequence of a viral protein.
 4. The ss-siRNA of any one of claims1-3, which is modified such that the ss-siRNA has increased in situ orin vivo stability as compared to a corresponding unmodified ss-siRNA. 5.The modified ss-siRNA of claim 4, which is modified by the substitutionof at least one nucleotide with a modified nucleotide.
 6. The modifiedss-siRNA of claim 5, wherein the modified nucleotide is a sugar-modifiednucleotide.
 7. The modified ss-siRNA of claim 6, wherein the modifiednucleotide has a 2′—OH replaced by a moiety selected from the groupconsisting of H, OR, R, halo, SH, SR¹, NH₂, NHR, NR₂ and CN, wherein Ris C₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
 8. Themodified ss-siRNA of claim 6, wherein the modified nucleotide is a 3′most nucleotide.
 9. The modified ss-siRNA of claim 8, wherein the 3′most nucleotide has a 2′—OH replaced by a moiety selected from the groupconsisting of H, OR, R, halo, SH, SR¹, NH₂, NHR, NR₂ and CN, wherein Ris C₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
 10. Themodified ss-siRNA of claim 8, wherein the 3′ most nucleotide has a 3′—OHreplaced by a moiety selected from the group consisting of H, OR, R,halo, SH, SR¹, NH₂, NHR, NR₂ and CN, wherein R is C₁-C₆ alkyl, alkenylor alkynyl and halo is F, Cl, Br or I.
 11. The modified ss-siRNA ofclaim 8, wherein the modified nucleotide is a backbone-modifiednucleotide.
 12. The modified ss-siRNA of claim 11, wherein thebackbone-modified nucleotide contains a phosphorothioate group.
 13. Thess-siRNA of any one of claims 1-3, comprising between about 10 and 50nucleotides.
 14. The ss-siRNA of any one of claims 1-3, comprisingbetween about 15 and 45 nucleotides.
 15. The ss-siRNA of any one ofclaims 1-3, comprising between about 19 and 40 nucleotides.
 16. Thess-siRNA of any one of claims 1-3 which is chemically synthesized.
 17. Atransgene that encodes the ss-siRNA of any one of claims 1-3.
 18. Acomposition comprising the ss-siRNA molecule of any one of claims 1-3and a pharmaceutically acceptable carrier.
 19. A method of activatingtarget-specific RNA interference (RNAi) in a cell comprising introducinginto said cell a single-stranded small interfering RNA molecule(ss-siRNA), wherein the sequence of said ss-siRNA molecule issufficiently complementary to a target mRNA sequence to directtarget-specific RNA interference (RNAi) and wherein the 5′ nucleotide is5′ phosphorylated or is capable of being 5′ phosphorylated in situ or invivo, said ss-siRNA being introduced in an amount sufficient fordegradation of the target mRNA to occur, thereby activatingtarget-specific RNAi in the cell.
 20. The method of claim 19, whereinthe ss-siRNA is introduced into the cell by contacting the cell with thess-siRNA.
 21. The method of claim 20, wherein the ss-siRNA is introducedinto the cell by contacting the cell with a composition comprising thess-siRNA and a lipophillic carrier.
 22. The method of claim 19, whereinthe ss-siRNA is introduced into the cell by transfecting or infectingthe cell with a vector comprising nucleic acid sequences capable ofproducing the ss-siRNA when transcribed in situ.
 23. The method of claim19, wherein the ss-siRNA is introduced into the cell by injecting intothe cell a vector comprising nucleic acid sequences capable of producingthe ss-siRNA when transcribed in situ.
 24. The method of claim 23,wherein the vector comprises transgene nucleic acid sequences.
 25. Themethod of claim 19, wherein the target mRNA specifies the amino acidsequence of a protein involved or predicted to be involved in a humandisease or disorder.
 26. A cell obtained by the method of claim
 19. 27.The cell of claim 26 which is of mammalian origin.
 28. The cell of claim26 which is of murine origin.
 29. The cell of claim 26 which is of humanorigin.
 30. The cell of claim 26, which is an embryonic stem cell. 31.An organism derived from the cell of claim
 30. 32. A method ofactivating target-specific RNA interference (RNAi) in an organismcomprising administering to said organism a single-stranded smallinterfering RNA molecule (ss-siRNA), wherein the sequence of saidss-siRNA molecule is sufficiently complementary to a target mRNAsequence to direct target-specific RNA interference (RNAi) and whereinthe 5′ nucleotide is 5′ phosphorylated or is capable of being 5′phosphorylated in situ or in vivo, said ss-siRNA being administered inan amount sufficient for degradation of the target mRNA to occur,thereby activating target-specific RNAi in the organism.
 33. The methodof claim 32, wherein the ss-siRNA is administered by an intravenous orintraperitoneal route.
 34. The method of claim 32, wherein the targetmRNA specifies the amino acid sequence of a protein involved orpredicted to be involved in a human disease or disorder.
 35. An organismobtained by the method of claim
 32. 36. The organism of claim 35 whichis of mammalian origin.
 37. The organism of claim 35 which is of murineorigin.
 38. The organism of claim 35 which is of human origin.
 39. Theorganism of any one of claims 35-38, wherein the target mRNA specifiesthe amino acid sequence of a protein involved or predicted to beinvolved in a human disease or disorder.
 40. The organism of any one ofclaims 35-38, wherein degradation of the target mRNA produces aloss-of-function phenotype.
 41. The method of claim 19-24 and 32-34,wherein degradation of the target mRNA is such that the proteinspecified by said target mRNA is decreased by at least 10%.
 42. A methodof treating a disease or disorder associated with the activity of aprotein specified by a target mRNA in a subject, comprisingadministering to said subject a single-stranded small interfering RNAmolecule (ss-siRNA), wherein the sequence of said ss-siRNA molecule issufficiently complementary to the target mRNA sequence to directtarget-specific RNA interference (RNAi) and wherein the 5′ nucleotide is5′ phosphorylated or is capable of being 5′ phosphorylated in situ or invivo, said ss-siRNA being administered in an amount sufficient fordegradation of the target mRNA to occur, thereby treating the disease ordisorder associated with the protein.
 43. A method for derivinginformation about the function of a gene in a cell or organismcomprising: (a) introducing into said cell or organism a single-strandedsmall interfering RNA molecule (ss-siRNA), wherein the sequence of saidss-siRNA molecule is sufficiently complementary to a target mRNAsequence to direct target-specific RNA interference (RNAi) and whereinthe 5′ nucleotide is 5′ phosphorylated or is capable of being 5′phosphorylated in situ or in vivo; (b) maintaining the cell or organismunder conditions such that target-specific RNAi can occur; (c)determining a characteristic or property of said cell or organism; and(d) comparing said characteristic or property to a suitable control, thecomparison yielding information about the function of the gene.
 44. Amethod of validating a candidate protein as a suitable target for drugdiscovery comprising: (a) introducing into a cell or organism asingle-stranded small interfering RNA molecule (ss-siRNA), wherein thesequence of said ss-siRNA molecule is sufficiently complementary to atarget mRNA sequence to direct target-specific RNA interference (RNAi)and wherein the 5′ nucleotide is 5′ phosphorylated or is capable ofbeing 5′ phosphorylated in situ or in vivo, said target mRNA specifyingthe amino acid sequence of the candidate protein; (b) maintaining thecell or organism under conditions such that target-specific RNAi canoccur; (c) determining a characteristic or property of said cell ororganism; and (d) comparing said characteristic or property to asuitable control, the comparison yielding information about whether thecandidate protein is a suitable target for drug discovery.
 45. A kitcomprising reagents for activating target-specific RNA interference(RNAi) in a cell or organism, said kit comprising: (a) an isolated,single-stranded small interfering (ss-siRNA) molecule, wherein thesequence of said ss-siRNA molecule is sufficiently complementary to atarget mRNA sequence to direct target-specific RNA interference (RNAi)and wherein the 5′ nucleotide is 5′ phosphorylated or is capable ofbeing 5′ phosphorylated in situ or in vivo; and (b) instructions foruse.